Method for drying water-borne materials

ABSTRACT

Provided is a method of isolating a bio-molecule from a water-borne mixture, the method comprising: contacting the water-borne mixture with dimethyl ether to form solid particles of the bio-molecule.

[0001] This application claims priority from U.S. ProvisionalApplication Serial No. 60/359,665, filed Feb. 25, 2002, he disclosure ofwhich is hereby incorporated by reference as if fully set forth herein.

[0002] The present invention relates to a method for isolating driedsolid particles from a water-borne mixture.

[0003] The recovery of a material dissolved or suspended in water is acommon need in industry, and removal of water by drying is common. Forexample, large quantities of food and milk are dried to facilitate longterm storage. Sterilized solutions or water suspensions ofpharmaceuticals are dried for subsequent ease in preparing exact dosageforms. Many chemicals including dyes and pigments are synthesized inaqueous solutions or suspensions, and are subsequently isolated bydrying. Typical drying methods include spray drying, belt drying, freezedrying (also known as lyophilization) and critical point drying. Theselection of the drying method in each case is dependent upon thedesired properties in the dried material such as stability,pharmaceutical activity, or particle size.

[0004] While useful in many applications, known drying methods are oftenunsatisfactory for drying water-borne mixtures containing bio-molecules(e.g., peptides, proteins, DNA, RNA, and polysaccharides). For example,human blood typically cannot be isolated by spray drying techniques dueto insufficient heat stability of some of the proteins containedtherein. Similarly, pharmaceutical proteins (e.g., growth factors,vaccines, hormones, monoclonal antibodies and many therapeutic proteins)cannot generally be spray-dried due to their tendency to denature underthe process conditions. Moreover, ultra-fine or microporous materialsfrom water-borne mixtures cannot generally be isolated by spray-drying,belt drying or tray drying if retention of ultra-fine size or porosityis desired in the isolated material. These drying techniques oftenresult in strong physical aggregation and severe pore collapse of theparticles due to the large surface tension forces active during thevaporization of water.

[0005] Techniques that include the use of supercritical or near criticalfluids have been utilized in alternative drying techniques. Thesetechniques generally include the use of carbon dioxide or other fluidsthat are immiscible in water, and they also generally use an organicsolvent, typically ethanol. Contact of the bio-molecule with suchorganic solvent-fluid-water mixtures often results in irreversibledegradation of fragile bio-molecules. This process can also causeunfolding or denaturation of proteins which can result in loss of theproteins' bioactivity.

[0006] To avoid these difficulties, critical point drying can be used toform particles that maintain the shape and structure of the substrate.Critical point drying processes include the steps of soaking awater-borne mixture with a water-miscible organic solvent, e.g.,acetone, ethanol; introducing carbon dioxide to the mixture to form acarbon-dioxide-solvent solution; raising the temperature of the systemto above the critical point temperature of the carbon-dioxide-solventsolution; and removing the now gaseous solution while maintaining thetemperature above the critical point temperature of the carbondioxide-solvent solution to isolate the dried particles. While criticalpoint drying achieves the formation of solid particles without porecollapse or strong physical aggregation of the particles, the entireprocess is a laborious one. In certain cases the technique can alsoresult in the denaturation of the bio-molecules and a drastic decreasein the bio-molecules' bioactivity.

SUMMARY OF THE INVENTION

[0007] In one embodiment, the invention relates to a method of isolatinga bio-molecule from a water-borne mixture. The method includes the stepof contacting the water-borne mixture with dimethyl ether to form solidparticles of the bio-molecule. Water-borne mixtures include aqueoussolutions, suspensions, emulsions, micro-emulsions and liposomessuspended in aqueous media. The water-borne mixture includes thebio-molecule and a solvent component.

[0008] In one embodiment, the method is conducted at a temperature of 0to 250° C., and the dimethyl ether is at a pressure range from P_(c) to10×P_(c), wherein P_(c) is the critical pressure of dimethyl ether.Preferably, the method is conducted at a temperature of 0 to 80° C., andthe dimethyl ether is at a pressure range from P_(c) to 2×P_(c).

[0009] In another embodiment, the method is conducted at a temperatureof 0 to 126.5° C. and the dimethyl ether is at a pressure range from 100psi to 2×P_(c). Preferably, the method is conducted at a temperature of0 to 60° C., and the dimethyl ether is at a pressure range from 200 psito P_(c).

[0010] In still another embodiment, the method is conducted is conductedat a temperature of 0 to 126.5° C. and the dimethyl ether is at apressure range from 50 psi to P_(c). Preferably, the method is conductedat a temperature of 0 to 60° C.

[0011] In one aspect of the method (the static method), the bio-moleculeis isolated from the water-borne mixture by introducing the dimethylether into a pressurized chamber containing the water-borne mixture. Inanother aspect of the method (the dynamic method), the bio-molecule isisolated by injecting the water-borne mixture into a stream of thedimethyl ether.

[0012] In one preferred embodiment, the isolated bio-molecule is aprotein. For example, the method of the invention can be used to isolatea hormone such as insulin. The method of the invention is also usefulfor isolating an enzyme (e.g., chymotrypsin) from a water-borne mixturewhich can be recovered with minimal, if any, loss of catalytic activity.

[0013] In another preferred embodiment, the isolated bio-molecule is apolynucleotide such as DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a depiction of one embodiment of an apparatus useful forconducting the invention method.

[0015]FIG. 2 is a depiction of another embodiment of an apparatus usefulfor conducting the invention method.

Definitions

[0016] The definitions of certain terms used herein are as follows:“bio-molecule” refers to peptides, proteins, polynucleotides andpolysaccharides. “water-borne mixture” refers to aqueous solutions,suspensions, emulsions, microemulsions and liposomes suspended inaqueous media.

DETAILED DESCRIPTION OF THE INVENTION

[0017] In accordance with the invention, Applicants have found a methodfor isolating a substance from a water-borne mixture containing thesubstance by contacting the water-borne mixture with compressed dimethylether to form solid particles of the substance. The invention provides arobust, scaleable, low-cost process for separating water from solidparticles while maintaining the product's desired characteristics. Themethod achieves the drying of particles without significant particleaggregation from water-borne mixtures, including solutions andsuspensions. The combination of these product characteristics and thecompatibility with water-soluble or water-dispersible substances makesthe process of the invention particularly desirable for bio-moleculessuch as polypeptides, proteins, polynucleotides and polysaccharides.

[0018] Applicants have found that compressed gaseous, liquid orsupercritical dimethyl ether is particularly advantageous for dryingparticles from water-borne mixtures using extraction techniques.Dimethyl ether (also known as methyl ether) is very soluble in water,and also dissolves water. This solubility is maintained along the entirevapor-pressure curve of dimethyl ether from about −5° C. to above itscritical temperature (Tc) of 126.9° C. While not being bound by theory,Applicants believe the drying of porous particles without pore collapseis achieved because dimethyl ether and water form a single phase duringthe extraction process. The formation of a single phase is beneficial inovercoming the difficulties that typically plague processes that entailvaporization of water from a two-phase system of liquid water and watervapor. Other commonly used fluid solvents such as carbon dioxide,nitrous oxide, hydrocarbons and xenon are insoluble in water, andtherefore do not achieve a single phase with water.

[0019] In one embodiment of the method, particles of desired substanceare dried by charging a water-borne mixture into a drying chamber andthen introducing gaseous dimethyl ether into the chamber. This staticmethod can be considered as a water-extraction method as the aqueoussolvent is extracted by the dimethyl ether leaving the dried particlesin the drying chamber.

[0020]FIG. 1 shows one embodiment of an apparatus that can be used forconducting the static water-extraction method described above. Theapparatus depicted in FIG. 1 includes a gas source housed in apressurized cylinder 1, a gas compressor 2, and a pressure resistantdrying chamber 6. The water-borne mixture containing the desiredsubstance is charged to the drying chamber 6. The dimethyl ether (gas orliquid) is taken from the pressure cylinder 1 and compressed by gas pump2. At a set pressure, monitored by pressure gauge 3, the dimethyl etheris fed through a needle valve 4 and introduced into the drying chamber6. The dimethyl ether extracts the aqueous solvent component leavingdried solid particles in the filter 5, located within the pressurizeddrying chamber itself or positioned directly at the inlet of thechamber. A pressure reduction valve 9, located at the outlet of thechamber controls the dimethyl ether flow rate. A liquid collector 10,traps the water and liquid solvent, while a thermocouple 7, and apressure gauge 8, monitors the conditions inside the chamber 6. A flowmeter 11 and dry test meter 12, positioned downstream of the liquidcollector 10, measure dimethyl ether flow rate and total volume,respectively. Dimethyl ether can be recycled using a closed loop toavoid dimethyl ether emission to the environment.

[0021] According to this static method, the size and morphology of theparticles produced from a solution are often determined in large part bythe properties of the solute substance itself, but certain processparameters can also often be manipulated to achieve the desiredmorphology and size of the dried particles. These parameters areapparent to those of ordinary skill in the art and include soluteconcentration, contact time in the drying chamber, temperature, pressureand nozzle size.

[0022] In another embodiment of the method, the water-borne mixturecontaining the desired substance, either in solution or suspension, issprayed through an atomization nozzle into an established stream ofdimethyl ether in a pressurized drying chamber. In the case of thisdynamic method, the dimethyl ether extracts the aqueous solventcomponent causing the desired substance to precipitate as dried solidparticles.

[0023]FIG. 2 shows an apparatus for conducting the dynamic methoddescribed above. In the apparatus, dimethyl ether, gas or liquid, isstored in pressurized cylinder 1. The fluid solvent exits cylinder 1 andis compressed by gas pump 2. At a predetermined pressure, monitored bypressure gauges 3 and 8, the fluid solvent traverses needle valve 4 andenters a pressure resistant drying chamber 6A. Once a desiredsupercritical solvent flow is established by adjusting valve 4, which ismonitored by flow meter 11, a water-borne mixture containing the desiredsubstance is fed from the solution reservoir 14 to the liquid gas pump15 via needle valve 9A. The water-borne mixture subsequently enters thegas stream via a nozzle 16, which is disposed within the drying chamber6A. The interaction between the supercritical fluid and aqueoussolution, through nozzle 16, facilitates nucleation. A filter 5 trapsthe solid, stable particles that are recrystallized or dried, and liquidcollector 10, traps the aqueous medium. A back pressure regulator 13,disposed downstream of drying chamber 6A, regulates the pressure insidethe chamber. A thermocouple 7 and pressure gauge 8 monitor theconditions inside chamber 6A. A flow meter 11 and dry test meter 12,disposed downstream of back pressure regulator 13 measure thesupercritical fluid's flow rate and total volume, respectively.

[0024] In this dynamic method, the rates of dimethyl ether addition tothe water-borne mixture addition can be altered to achieve the desiredparticle size and shape. Other parameters, apparent to those of ordinaryskill in the art, can also be manipulated such as contact time in thedrying chamber, temperature, pressure and nozzle size.

[0025] The water-borne mixture includes the desired substance and asolvent component, that is composed in large part by water. Generally,the solvent component contains a significant proportion of water;typically at least 20%, preferably at least 40% by volume water, andmore preferably at least 80% by volume water. In addition to water, thesolvent component can also include other water-miscible solvents inmixture with water including but not limited to lower alcohols (e.g.,methanol, ethanol, n-propanol, isopropanol), lower ketones (e.g.,acetone) acetonitrile, methyl sulfoxide, dimethylformamide, ethers(e.g., diethyl ether), lower esters (e.g., ethyl acetate) and mixturesthereof.

[0026] The dried product from the process can be used for long-termstorage of edible and potable foodstuffs, for pharmaceutical purposesfor both human and veterinary applications (therapeutic, prophylacticand diagnostic purposes), for cosmetic applications or in otherapplications where a water-soluble or water-dispersible substrate is tobe comminuted. In instances where the substances are used forpharmaceutical purposes, the dried substance can be administered aloneor it can be further processed in combination with known excipients inconventional pharmaceutical formulations. These pharmaceutical agents orformulation thereof can be administered by oral, nasal, rectal, buccal,intraocular, pulmonary, transdermal, or parenteral routes.

[0027] In a preferred embodiment of the invention, the substance to bedried includes a bio-molecule selected from the group consisting ofpeptides, proteins, polynucleotides (e.g., DNA, RNA) and polysaccharides(e.g., heparin). For instance, the methods of the invention are usefulfor drying milk which is an aqueous emulsion containing proteins as wellas lipids. The methods of the invention are particularly useful fordrying protein particles, as the effects of the drying process on thetertiary structures of the proteins are minimal. As a result, thebiological activity in the dried protein particles are maintained.Similarly, the structural integrity of the polynucleotides are alsomaintained by the drying methods of the invention. In one preferredembodiment, the bio-molecules are non-acid labile substances.

[0028] The method can be conducted at operating temperatures of about 0°C. to 250° C., preferably at 0° C. to 60° C. The pressure of the dryingcolumn is preferably maintained at about 50 to about 3,000 psi, morepreferably about 100 to about 700 psi.

[0029] In one preferred embodiment of the method, the dimethyl ether isat a pressure equal to P_(c) to 10×P_(c) , wherein P_(c) is the criticalpressure of dimethyl ether (53.7 bar). More preferably, the dimethylether is at a pressure equal to of P_(c) to 2×P_(c). In this embodiment,the method is preferably conducted at a temperature of 0 to 250° C.,more preferably at a temperature of 0 to 80° C.

[0030] In another preferred embodiment of the method, the dimethyl etheris at a pressure of 100 psi to 2×P_(c). More preferably, the dimethylether is at a pressure of 200 psi to P_(c). In this embodiment, themethod is preferably conducted at a temperature of 0 to 126.5° C., morepreferably at a temperature of 0 to 60° C.

[0031] In another preferred embodiment of the method, the dimethyl etheris at a pressure of 50 psi to P_(c). In this embodiment, the method ispreferably conducted at a temperature of 0 to 126.5° C., more preferablyat a temperature of 0 to 60° C.

[0032] The invention also provides a method for removing impurities froma desired substance. The impurities are extracted from the desiredsubstance by the compressed dimethyl ether during the process of dryingas described above, either according to the static or the dynamicmethod.

[0033] The invention also provides a method of removing infectiousagents from a desired substance, as compressed dimethyl ether is aneffective biocide against bacteria, fungi and viruses.

[0034] The following examples further illustrate the present invention,but of course, should not be construed as in any way limiting its scope.

EXAMPLE 1 Preparation of Dried Skim Milk

[0035] Skim milk, which contains both soluble and suspended protein wasloaded into the solution reservoir. The dimethyl ether was introducedinto the pressurized drying vessel at 25° C. and 1000 psi. A gas flowrate of 145 g/min of dimethyl ether was established. The milk wasintroduced into the dimethyl ether stream in the pressurized vessel at avolumetric flow rate of 3-6 mL/min. with an orifice of 50 μm. Dried,stable, free flowing particles of 1-2 μm were collected on the filter.

EXAMPLE 2 Preparation of Dried Silica

[0036] A 20 wt. % suspension of 200 nm size silica gel in water wascharged to a pressure drying vessel and the vessel was sealed. Dimethylether was supplied to the drying chamber at 130° C. and 1500 psi.Dimethyl ether extracted the suspension at a ratio of 5 g of dimethylether/1 g of suspension. The silica was recovered as a free flowingpowder consisting of 175-375 nm spheres.

EXAMPLE 3 Preparation of Dried Chymotrypsin

[0037] Fragments of chymotrypsin (20 to 80 μm in diameter) weredissolved in water to produce a 0.5% by weight aqueous solution. Thesolution was then charged into the solution reservoir. The dimethylether was introduced into the pressurized drying/particle formationvessel at 24° C. and 2500 psi. When the gas flow rate of about 100 SLPM(standard liter per minute) was established, the chymotrypsin solutionwas pumped into the system at a volumetric flow rate of 1.6 mL/min. Theprotein solution was atomized into the dimethyl ether gas stream via a63 μm nozzle. The dissolved protein was precipitated from the solutionand dried by dissolution of the water in the dimethyl ether. Dry, stablefree-flowing protein powder was collected in the filter. Scanningelectron microscopy revealed the processed protein particles were mostlyspherical and in the range of 0.4 to 0.8 μm in diameter. Chymotrypsinwas analyzed for activity according to the standard method specified inThe United States Pharmacopeia/The National Formulary (USP 25/NF 20),25^(th) Edition, pages 416-417. Analysis indicated that 95% of activitywas maintained. Moisture analysis indicated that the residual moistureof the processed powder was 7.4%, which was the same as that of theunprocessed raw (starting) material.

EXAMPLE 4 Preparation of Dried Bovine Serum Albumin

[0038] A bovine serum albumin (BSA) aqueous solution containing 2.0% byweight of albumin was prepared and charged into the solution reservoir.The dimethyl ether was introduced into the pressurized drying/particleformation vessel at 40° C. and 2300 psi. When the gas flow rate of about100 SLPM was established, the BSA solution was pumped into the system ata volumetric flow rate of 1.6 mL/min. The protein solution was atomizedinto the dimethyl ether gas stream via a 63 μm nozzle. The dissolvedprotein was precipitated from the solution and dried by dissolution ofthe water in the dimethyl ether. Dry, stable free-flowing protein powderwas collected in the filter. Scanning electron microscopy revealed theprocessed protein particles were spherical and in the submicron range.Fourier transform infrared (FTIR) analysis was used to characterize theprotein structure. Particle size measurement using laser diffractiontechnique shows that the processed particles had a median diameter of2.8 μm. The secondary structure of BSA was quantified by Gaussiancurve-fitting of the resolution-enhanced amide I band using a Magna-IRsystem (Nicolet, Wis., USA). This analysis indicated that the structuralactivity of the protein was maintained. Moisture analysis indicates thatthe residual moisture of the processed powder was 7.2%, which was aboutthe same as that of the unprocessed raw (starting) material.

EXAMPLE 5 Preparation of Dried Bovine Insulin

[0039] A bovine insulin (zinc) aqueous solution containing 1.5% byweight of insulin was prepared in 0.01 M hydrochloride (HCl) solutionand charged into the solution reservoir. Dimethyl ether was introducedinto the pressurized drying/particle formation vessel at 37° C. and 2300psi. When the gas flow rate of about 100 SLPM was established, theinsulin solution was pumped into the system at a volumetric flow rate of1.6 mL/min. The protein solution was pulverized into the dimethyl ethergas stream via a 63 μm nozzle. The dissolved protein was precipitatedfrom the solution and dried by dissolution of the water in the dimethylether. Dry, stable free-flowing protein powder was collected in thefilter. Scanning electron microscopy revealed the processed proteinparticles were mostly spherical and in the 1-4 μm range. Particle sizemeasurement using laser diffraction technique shows that the processedparticles have a median diameter of 2.8 μm. Analysis indicated that 100%of activity was maintained. Moisture analysis indicated that theresidual moisture of the processed powder was 8.4%, which was about thesame as that of the dry unprocessed raw (starting) material.

EXAMPLE 6 Preparation of Dried DNA

[0040] Unprocessed DNA material, composed of chunky strands, wasdissolved in water to form an aqueous solution of DNA containing 0.5% byweight of DNA. The solution was charged into the solution reservoir. Thedimethyl ether was introduced into the pressurized drying/particleformation vessel at 37° C. and 2500 psi. When a gas flow rate of about100 SLPM was established, the DNA solution was pumped into the system ata volumetric flow rate of 1.6 mL/min. The DNA solution was atomized intothe dimethyl ether gas stream via a 63 μm nozzle. The dissolved DNA wasprecipitated from the solution and dried by dissolution of the water indimethyl ether. Dry, stable free-flowing powder was collected in thefilter. Scanning electron microscopy revealed the processed proteinparticles were spherical and in the range of 0.5 to 1.0 μm in diameter.

What is claimed:
 1. A method of isolating a bio-molecule from awater-borne mixture, the method comprising: contacting the water-bornemixture with dimethyl ether to form solid particles of the bio-molecule.2. The method as recited in claim 1, wherein the water-borne mixturecomprises the bio-molecule and a solvent component.
 3. The method asrecited in claim 1, wherein the water-borne mixture is a suspension. 4.The method as recited in claim 1, wherein the contacting is conducted ata temperature of 0 to 250° C. and the dimethyl ether is at a pressurerange from P_(c) to 10×P_(c), wherein P_(c) is the critical pressure ofdimethyl ether.
 5. The method of claim 4, wherein the contacting isconducted at a temperature of 0 to 80° C. and the dimethyl ether is at apressure range from P_(c) to 2×P_(c).
 6. The method as recited in claim1, wherein the contacting is conducted at a temperature of 0 to 126.5°C. and the dimethyl ether is at a pressure range from 100 psi to2×P_(c), wherein P_(c) is the critical pressure of dimethyl ether. 7.The method as recited in claim 6 wherein the contacting is conducted ata temperature of 0 to 60° C. and the dimethyl ether is at a pressurerange from 200 psi to P_(c).
 8. The method as recited in claim 1,wherein the contacting is conducted at a temperature of 0 to 126.5° C.and the dimethyl ether is at a pressure range from 50 psi to P_(c),wherein P_(c) is the critical pressure of dimethyl ether.
 9. The methodas recited in claim 8 wherein the contacting is conducted at atemperature of 0 to 60° C.
 10. The method as recited in claim 1, whereinthe dimethyl ether is introduced into a pressurized chamber containingthe water-borne mixture.
 11. The method as recited in claim 1, whereinthe water-borne mixture is injected into a stream of the dimethyl ether.12. The method as recited in claim 1, wherein the bio-molecule is aprotein.
 13. The method as recited in claim 12, wherein the protein is ahormone.
 14. The method as recited in claim 13, wherein the hormone isinsulin.
 15. The method as recited in claim 12, wherein the protein isan enzyme.
 16. The method as recited in claim 1, wherein thebio-molecule is a polynucleotide.
 17. The method as recited in claim 16,wherein the polynucleotide is DNA.